Crosslinked protein with acid anhydride as a ruminant feed material

ABSTRACT

Improved protein feed material for ruminants which is resistant to digestive breakdown in the rumen but not in the abomasum and/or intestines which comprises the reaction product of a protein-containing feed material and an organic acid anhydride capable of crosslinking protein. Exemplary of such organic acid anhydrides is maleic anhydride.

United States Patent Miller 1 1March 13, 1973 1 CROSSLINKEI) PROTEINWITH ACID 3,541,204 11 1970 Sibbald et a1. ..99 2 R ANHYDRIDE AS ARUMINANT FEED FOREIGN PATENTS OR APPLICATIONS MATERIAL [75] Inventor:Robert E. Miller, Ballwin, Mo. 12381158 4/1967 Germany [73] Assignee:Monsanto Company, St. Louis, Mo. OTHER PUBLICATlONS 22 il Oct 27 1970 P..l. G. Butler et al., Biochemistry Journal, Vol. 103,

p. 78P-79P, 1967. 211 App1.No.: 84,471

Primary ExaminerNorman Yudkoff 52 11.5. c1 ..424/177, 99/2 R, 99/14,Assistant Examinercuflis Ribando 2 0 R, 260/119, 260/1235Attorney-Lyhden N. Goodwin, Neal E. Willis and 51 Int. 01...... ..A23kl/l8 James Williams, [58] Field of Search ..99/2 R, 2 N, 14, 17, 18, 7,

99/2 ND;260/112 R, 112 B, 112 T, 112 G, 1 1 ABSTRACT 117, 119, 123, 121,123.5; 424/177 Improved protein feed material for ruminants which isresistant to digestive breakdown in the rumen but not [56] ReferencesC'ted in the abomasum and/or intestines which comprises UNITED STATESPATENTS the reaction product of a protein-containing feed mater1a1 andan orgamc acld anhydnde capable of 3,108,995 10/1963 Tourtellotte et a1...260/112 R crosslinking protein. Exemplary of such organic acid2,518,666 8/1950 Damschroder et a1. ..260/123 anh drides is maleicanhydride. 2,009,029 7/1935 Sly ..260/1l9 2,932,589 4/1960 Meyer et al..260/666 R 4 Claims, No Drawings CROSSLINKED PROTEIN WITH ACIDANIIYDRIDE AS A RUMINANT FEED MATERIAL BACKGROUND OF THE INVENTION 1.Field of the Invention This invention relates to a method for improvingthe feed utilization of ruminant animals. In a particular aspect thisinvention relates to a method for improving protein utilization inruminant animals. In a further aspect this invention relates to modifiedprotein feed compositions useful in ruminant nutrition which areresistant to digestive attack in the fluid medium of the rumen.

2. Description of the Prior Art The digestive system of the ruminantanimal (cattle, sheep, bison, camels, etc.) is designed to permitefficient use of coarse, fibrous foodstuffs. Because of its particularstructure and nature, however, the ruminants digestive system isinefficient in obtaining nutritional value from protein materials.Principally for this reason it is common practice in ruminant nutritionto supplement the diet of the animal with added protein. Thesupplemental protein serves to increase the rate of growth of the animaland in the case of sheep promotes wool growth.

The rumen, the largest of the four stomach compartments of the animal,serves as an important location for digestive breakdown of ingestedfoodstuffs chiefly through the action of microorganisms present therein.However, absorption of most nutrients for metabolic purposes does notoccur in the rumen but takes place further along in the alimentarytract, principally in the abomasum and intestines. ingested food istypically retained in the rumen for from about 12-30 hours during whichtime it is subject to digestive breakdown by the microorganisms and bythe rumen fluid. Much ingested protein material is broken down in therumen to soluble peptides and amino acids. In turn much of thesepeptides and amino acids are utilized by the microorganisms present inthe rumen fluid thereby removing them as a source of nutrition for thehost animal.

Because of the desirability as indicated above of avoiding proteinbreakdown in the rumen in order to permit absorption in the abomasum andintestines it has been suggested that nutrient protein-containingmaterials fed to ruminants be treated so as to permit passage withoutdigestive breakdown through the rumen to the abomasum. Suggestedprocedures have included coating the protein material, for example, withfats and vegetable oils, heat treatment of the protein material andreaction of the protein material with formaldehyde. In any event thetreated material must be resistant to digestive breakdown in the rumenfluid, which is a fluid buffered at about pH6-7 by phosphate-bicarbonatefrom saliva and carbon dioxide, but subject to breakdown in the acidmedium of the fluid of the abomasum which has a pH, due principally tohydrochloric acid secretion, of about 2-4.

OBJECTS It is an object of the present invention to provide a method forimproving the feed utilization of ruminant animals.

It is a further object of the present invention to provide a method forimproving the protein utilization of ruminant animals whereby proteinpasses through the rumen without substantial digestive breakdown.

Other objects and advantages of the present invention will be apparentfrom the specification and appended claims.

SUMMARY OF THE INVENTION It has been found in accordance with thepresent invention that the protein utilization of the ruminant animal isimproved by feeding the animal a reaction product of aprotein-containing nutrient material and 0 an organic acid anhydridemonomer capable of cross- DETAILED DESCRIPTION The protein-containingreaction product used in the method of the present invention is thereaction product of a protein-containing nutrient feed material and anorganic acid anhydride monomer. Suitable organic acid anhydride monomersfor use in the method of the present invention include maleic anhydride,itaconic anhydride, citraconic anhydride, succinic anhydride, andphthalic anhydride, etc.

The protein-containing nutrient material can be from any suitable sourceincluding animal, plant, or synthetic sources such as, for example,silage, grains,

nuts, chaffs, casein, soy bean meal, fish meal, peanut meal, beefscraps, pork scraps, linseed meal, milk solids, etc.

The useful reaction products of the present invention are prepared bythe interaction of organic acid anhydride monomer and protein nutrientmaterial by any suitable procedure. The reaction is readily carried outin a suitable solvent medium such as water, acetic acid, aqueouspotassium hydroxide, and potassium acetate. The reaction may be carriedout at any suitable temperature, however, elevated temperatures aboveabout C particularly for extended periods should be avoided to minimizedegradation of protein material. Temperatures in the range of from about10C to about 40C are typically employed with room temperature being bothsuitable and practical. The reaction is preferably carried out simply byforming a slurry of the reactants in the solvent medium and agitating,as by stirring, the slurry thereby to permit sufficient reaction of theprotein with the organic acid anhydride. The thus treated protein isthen filtered, washed with water and dried as by oven drying, drumdrying, or simple evaporation to recover the modified protein nutrientmaterial. The organic acid anhydrides of use in the present inventionare those which cross-link the protein to form a complex which is stableunder the pH conditions of the rumen but unstable under the conditionsof the abomasum and intestines. It is believed the crosslinks are formedin the terminal a-amino groups of various peptide chains or in thee-amino groups of lysineor between the amide groups of asparagine andglutamine or between the guanidyl groups of arginine or between anycombination of these or other suitable groups available forcrosslinking.

It is important in order to insure operability of the present inventionthat the amount of organic acid anhydride incorporated into the proteinbe sufficient to prevent digestive breakdown to soluble peptides andamino acids in the rumen but insufficient to prevent digestive breakdownto soluble peptides and amino acids in the abomasum and intestines. Thisamount will, of course, vary and will depend among other things on theparticular protein material, the particular organic acid anhydride ofchoice, the pH of the solvent of reaction, time and temperature ofreaction, the species and age of the animal, and the total makeup of theanimal diet. Typically an amount in the range of from about 0.005 toabout 0.1 mole of organic acid anhydride for each gram of proteincontained in the protein material is employed with amounts in the rangeof from about 0.01 to about 0.05 mole being generally preferred.

It is to be understood that the modified ruminant protein feed of thepresent invention can be fed separately to the animal or it can be usedfor incorporation in other ruminant feed materials. Illustrative ofruminant feed materials in which the protein material of the presentinvention may be incorporated are soy bean meal, ground corn, hay,straw, cotton seed hulls, cotton mill waste, feed pulp, silage, oats,barley, cereal, brans, cereal middlings and combinations thereof. Ifdesired other components, for example, minerals, such as bone meal,salt, and trace minerals, antibiotics and vitamins may be included inthe animal feed ration.

The following examples illustrate the effectiveness of organic acidanhydride compositions useful in the present invention in protectingprotein-containing material from digestion in the fluid of the rumenwhile permitting digestion in the fluids of the abomasum and intestines.The small scale in vitro experiments shown in the examples simulateconditions existing in the rumen, in the abomasum and in the intestinesthereby permitting the study of treated protein without the use of thelive animal and large quantities of feed materials. It is understoodthat the examples are presented for the purpose of illustration only andthe invention is not limited to the compositions or methods showntherein.

EXAMPLE I Preparation of Treated Protein Casein (8.0 grams) was added toa solution of maleic anhydride (0.081 gram) in aqueous acetic acid (50milliliters). The resulting mixture was stirred for about 1 hour at roomtemperature to permit reaction of the maleic anhydride monomer withcasein. The reaction product was then filtered, washed copiously withwater and dried, to obtain protein product containing 0.0101 mole ofmaleic anhydride per gram of casein. Rumen Digestion Test To millilitersof rumen fluid from fasted sheep contained in a 50 milliliter glassflask was added [0 milliliters of a buffered solution of the followingcomposition.

Buffer Solution in Grams Per Liter Namro, 0.316

The resulting mixture was adjusted to pH6.8 (4N HCl). To the bufferedrumen fluid was added milligrams of the maleic anhydride treated proteinprepared above. The flask was then purged with nitrogen, stoppered(pressure release valve) and heated at 38C on a water shaker bath.Protection of protein from digestion was determined by ammoniaproduction with a lower amount of ammonia production indicating a loweramount of digestion of protein. Ammonia production was determined after6 hours and after 24 hours with results being presented in Table l.Abomasum Digestion Test Gastric Fluid was prepared as follows: NaCl (2grams) was dissolved in sufficient water to give a total volume of 950milliliters. Pepsin (3.2 grams) was added thereto. Concentratedhydrochloric acid (7 milliliters) was added to the resulting medium andthe pH of the medium was then adjusted to 2.0 with aqueous sodiumhydroxide.

To a glass flask containing 20 milliliters of the gastric fluid wereadded 60 milligrams of maleic anhydride treated casein prepared above.The glass flask containing the resulting mixture was stoppered withpressure release valves and heated at 38C on a water shaker bath for 2hours. Digestion of protein was then determined by ammonia analysis, thegreater amount of ammonia produced the greater the amount of proteindigested. The results are given in Table l.

Intestine Digestion Test Intestinal fluid was prepared as follows: NaCl(2 grams) was dissolved in sufficient water to give a total volume of950 milliliters. Pepsin (3.2 grams) was added thereto. Concentratedhydrochloric acid (7 milliliters) was added to the medium and the pH ofthe medium was then adjusted to 7.0 with 0.1 N sodium hydroxide.Pancreatin (10 milligrams per milliliter of medium) was added to theresulting medium.

To a glass flask containing 20 milliliters of the intestinal fluid wereadded 60 milligrams of maleic anhydride treated casein prepared above.The glass flask was stoppered with pressure release valves and heated at38C on a water shaker bath, for the prescribed period of time. Digestionof protein was determined by ammonia analysis, the greater amount ofammonia produced the greater amount of protein digested. The results aregiven in Table l.

EXAMPLES 2-3 Following the general procedures and tests of Example 1organic acid anhydride monomer treated casein samples were prepared andtested. The results are given in Table 1.

In the same manner other protein-containing materials may be reactedwith organic acid anhydride monomers to protect the protein materialfrom digestive breakdown in the rumen while permitting its digestion inthe abomasum.

EXAMPLE 4 Phthalic anhydride treated casein containing 0.05 molephthalic anhydride per gram of protein is resistant to digestion in thefluid of the rumen but is readily digested in the fluid of the abomasum.

EXAMPLE 5 Itaconic anhydride treated casein containing 0.05 moleitaconic anhydride per gram of casein is resistant to digestion in therumen but is readily digested in the abomasum.

Since many embodiments of this invention may be made and since manychanges may be made in the embodiments described, the foregoing is to beinterpreted as illustrative only and the invention is defined by the 0claims appended hereto.

TABLE 1 Percent total protein digested Ammonia N Ammonia N aboma-Ammonia N Moles anhyrumeu sum intestine Example Solvent o1 dride gramnumber Anhydntle preparation casein 6 hrs. 24 hrs. 2 hrs. 4 hrs. 20 hrs.

1 Maleic anhydride. Acetic acid 0.0101 20. 5 12.0 67. 8 73 82 2 d0 Water0.0101 77 64.3 88 3 ..do Formic acid"... 0. 0016 66 50.8

Due at least in part to decomposition of rumen microorganisms.

2. The method of claim 1 wherein the organic acid anhydride is maleicanhydride.

3. The method of claim 1 wherein the organic acid anhydride is phthalicanhydride.

4. The method of claim 1 wherein the amount of or- 30 ganic acidanhydride is in the range of from about 0.01

to about 0.05 mole per gram of protein.

l l i i

1. A method for improving the protein utilization of ruminant animalswhich comprises feeding the ruminant animal the reaction product of aprotein-containing nutrient feed material selected from the groupconsisting of silage, grains, nuts, chaffs, casein, soybean meal, fishmeal, peanut meal, beef scraps, port scraps, linseed meal and milksolids and an organic acid anhydride monomer to crosslink the protein,the monomer being selected from the group consisting of maleicanhydride, itaconic anhydride, citraconic anhydride, succinic anhydrideand phthalic anhydride, the amount of organic acid anhydride monomer insaid reaction product being in the range of from about 0.005, to about0.1 mole per gram of protein, said amount being sufficient to rendersaid feed material resistant to digestive breakdown in the fluid of therumen but insufficient to prevent substantial digestive breakdown in thefluids of the abomasum and of the intestines.
 2. The method of claim 1wherein the organic acid anhydride is maleic anhydride.
 3. The method ofclaim 1 wherein the organic acid anhydride is phthalic anhydride.